Pharmaceutical compositions of O-nitro compounds

ABSTRACT

The present invention provides O-nitro compounds, pharmaceutical compositions of O-nitro compounds and methods of using O-nitro compounds and/or pharmaceutical compositions thereof to treat or prevent diseases or disorders characterized by abnormal cell proliferation, such as cancer, inflammation, cardiovascular disease and autoimmune disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/502,974, filed Aug. 11, 2006, now abandoned, which application claimspriority to U.S. Provisional Application Ser. No. 60/707,896, filed Aug.12, 2005, each of which is hereby incorporated herein by this referencein its entirety.

TECHNICAL FIELD

The present invention relates generally to pharmaceutical compositionsof O-nitro compounds and methods of using 0-nitro compounds andpharmaceutical compositions thereof to treat or prevent diseasescharacterized by abnormal cell proliferation such as cancer.

BACKGROUND OF THE INVENTION

Abnormal cell proliferation is a characteristic symptom of cancer.Further, abnormal cell proliferation has been implicated in numerousother diseases (e.g., cardiovascular diseases, inflammatory diseasessuch as rheumatoid arthritis, diabetic retinopathy, etc.). Although manymethods for treating or preventing aberrant cell proliferation have beendeveloped, a significant problem with most existing therapies isselectively distinguishing between normal and abnormal cellproliferation.

Radiotherapy is one promising approach to selectively targeting abnormalcell proliferation. A number of different radiosensitizers have beendescribed in the art and include thiols, nitroimidazoles and metaltexaphyrin compounds (see, e.g., Rosenthal et al., Clin. Cancer Res.,1999, 739). Significant problems with existing radiosensitizationapproaches are (1) the formation of toxic byproducts derived from theradiosensitizers, which has limited their usefulness in cancer therapy;and (2) achieving sufficiently high density of free radicals to beefficacious under dose-limiting toxicity.

Another popular approach to selectively targeting abnormal cellproliferation is treatment with bioreductive compounds, which areselectively activated in a reducing environment. Since many cancerstypically contain regions of low oxygen tension (i.e., hypoxia),compounds with low redox potentials (i.e., bioreductive compounds) maybe selectively activated in the reducing environment of tumor cellswithout external activation.

Accordingly, new compounds are required to fully explore treating orpreventing abnormal cell proliferation. These new compounds may haveradiotherapeutic activity or bioreductive activity. Such compounds maybe effective in treating or preventing various diseases associated withabnormal cell proliferation such as cancer without forming toxicbyproducts.

SUMMARY OF THE INVENTION

The present invention satisfies this and other needs by providingO-nitro compounds, pharmaceutical compositions of O-nitro compounds andmethods of using O-nitro compounds or pharmaceutical compositionsthereof to treat or prevent diseases associated with abnormal cellproliferation.

In a first aspect, the present invention provides methods for treatingor preventing diseases or disorders characterized by abnormal cellproliferation. The methods generally involve administering to a patientin need of such treatment or prevention a therapeutically effectiveamount of an O-nitro compound or a pharmaceutically acceptable salt,hydrate, solvate or N-oxide thereof.

In a second aspect, the present invention provides pharmaceuticalcompositions of O-nitro compounds. The pharmaceutical compositionsgenerally comprise one or more O-nitro compounds, pharmaceuticallyacceptable salts, hydrates, solvates or N-oxides thereof, and apharmaceutically acceptable vehicle. The choice of vehicle will dependupon, among other factors, the desired mode of administration.

In a third aspect, the current invention provides pharmaceuticalcompositions for treating or preventing diseases or disorderscharacterized by abnormal cell proliferation. The methods generallyinvolve administering to a patient in need of such treatment orprevention a therapeutically effective amount of a pharmaceuticalcomposition comprising an O-nitro compound or a pharmaceuticallyacceptable salt, hydrate, solvate or N-oxide thereof and apharmaceutically acceptable vehicle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a possible mechanism of chemosensitization for O—NO₂compounds;

FIG. 2 illustrates the chemosensitization of glycidyl nitrate (“GLYN”)in combination with cis-platin on SCC VII tumor growth;

FIG. 3 illustrates radiation sensitization effects of GLYN and SG(sodium glycididazole) in SCC VII tumors;

FIG. 4 a illustrates that nitric oxide generated in tumor cells dependedon the dosage of GLYN;

FIG. 4 b illustrates the nitric oxide concentration in SCC VII cells asa function of time after exposure of SCC VII tumor cells to GLYN;

FIGS. 5 a-5 d illustrate the concentration of nitric oxide in SCC VIItumor cells after irradiation at ten minutes, one hour, two hours andsix hours, respectively.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl,” by itself or as part of another substituent, refers to asaturated or unsaturated, branched, straight-chain or cyclic monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane, alkene or alkyne. Typical alkylgroups include, but are not limited to: methyl; ethyls such as ethanyl,ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl,cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), cycloprop-1-en-1-yl, cycloprop-2-en-1-yl, prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

The term “alkyl” is specifically intended to include groups having anydegree or level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds and groupshaving mixtures of single, double and triple carbon-carbon bonds. Wherea specific level of saturation is intended, the expressions “alkanyl,”“alkenyl,” and “alkynyl” are used. In some embodiments, an alkyl groupcomprises from 1 to 20 carbon atoms. In other embodiments, an alkylgroup comprises 1 to 10 carbon atoms. In still other embodiments, analkyl group comprises from 1 to 6 carbon atoms.

“Alkanyl,” by itself or as part of another substituent, refers to asaturated branched, straight-chain or cyclic alkyl radical derived bythe removal of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to:methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkenyl,” by itself or as part of another substituent, refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkene. The groupmay be in either the cis or trans conformation about the double bond(s).Typical alkenyl groups include, but are not limited to: ethenyl;propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl, cycloprop-2-en-1-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,cyclobuta-1,3-dien-1-yl, etc., and the like.

“Alkynyl,” by itself or as part of another substituent, refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkyne. Typicalalkynyl groups include, but are not limited to: ethynyl; propynyls suchas prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc., and the like.

“Adamantyl,” by itself or as part of another substituent, refers tohydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of adamantane.

“Cycloalkyl,” by itself or as part of another substituent, refers to asaturated or unsaturated cyclic alkyl radical derived by the removal ofone hydrogen atom from a single carbon atom. Where a specific level ofsaturation is intended, the nomenclature “cycloalkanyl” or“cycloalkenyl” is used. Typical cycloalkyl groups include, but are notlimited to, groups derived from cyclopropane, cyclobutane, cyclopentane,cyclohexane, and the like. In some embodiments, the cycloalkyl group is(C₃-C₁₀) cycloalkyl. In other embodiments, the cycloalkyl group is(C₃-C₇) cycloalkyl.

“Cycloheteroalkyl,” by itself or as part of another substituent, refersto a saturated or unsaturated cyclic alkyl radical in which one or morecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom. Typical heteroatoms toreplace the carbon atom(s) include, but are not limited to, N, P, O, S,Si, etc. Where a specific level of saturation is intended, thenomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl” is used.Typical cycloheteroalkyl groups include, but are not limited to, groupsderived from epoxides, azirines, thiiranes, imidazolidine, morpholine,piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine and thelike.

“Fused cycloalkyl,” by itself or as part of another substituent, refersto a saturated or unsaturated fused cyclic alkyl radical of the formm.n.0 alkyl, where m and n are integers greater than 1, derived by theremoval of one hydrogen atom from a single carbon atom of a parent fusedcycloalkyl compound. Typical fused cycloalkyl groups include, but arenot limited to, 4.2.0 octane, 4.1.0 heptane, 3.2.0 heptane, 3.1.0hexane. In some embodiments, the fused cycloalkyl group is (C₃-C₁₀)fused cycloalkyl.

“Fused cycloheteroalkyl,” by itself or as part of another substituent,refers to a saturated or unsaturated fused cycloalkyl radical in whichone or more carbon atoms (and any associated hydrogen atoms) areindependently replaced with the same or different heteroatom. In someembodiments, the fused cycloheteroalkyl group is (C₃-C₁₀) fusedcycloheteroalkyl.

“Cubyl,” by itself or as part of another substituent, refers tohydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of cubane.

“Heteroalkyl, heteroalkanyl, heteroalkenyl and heteroalkynyl,” bythemselves or as part of another substituent, refer to alkyl, alkanyl,alkenyl and alkynyl groups, respectively, in which one or more of thecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatomic groups. Typicalheteroatomic groups that can be included in these groups include, butare not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—, —NR³⁴R³⁵—, ═N—N═,—N═N—, —N═N—NR³⁶R³⁷, —PR³⁸—, —P(O)₂—, —POR³⁹—, —O—P(O)₂—, —SO—, —SO₂—,—SnR⁴⁰R⁴¹— and the like, where R³⁴, R³⁵, R³⁶, R³⁷, R³⁸, R³⁹, R⁴⁰ and R⁴¹are independently hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl or substituted heteroarylalkyl.

“Pharmaceutically acceptable salt” refers to a salt of an O-nitrocompound, which is pharmaceutically acceptable and possesses the desiredpharmacological activity of the parent compound. Such salts: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, t-butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid,salicylic acid, stearic acid, muconic acid and the like; or (2) saltsformed when an acidic proton present in the parent compound is replacedby an ammonium ion, a metal ion, e.g., an alkali metal ion (e.g., sodiumor potassium), an alkaline earth ion (e.g., calcium or magnesium), or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, N-methylglucamine, morpholine,piperidine, dimethylamine, diethylamine and the like. Also included aresalts of amino acids such as arginates and the like, and salts oforganic acids like glucurmic or galactunoric acids and the like.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which an O-nitro compound is administered.

“Patient” includes humans and other mammals.

“Preventing” or “prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a patient that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease).

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Typical substituents include, but are not limited to -M, —R⁶⁰, —O⁻, ═O,—OR⁶⁰, —SR⁶⁰, —S⁻, ═S, —NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO,—NO₂, —ONO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R⁶⁰, —OS(O₂)O⁻,—OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —C(S)OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹,—NR⁶²C(S)NR⁶⁰R⁶¹, —NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹, where M isindependently a halogen; R⁶⁰, R⁶¹, R⁶² and R⁶³ are independentlyhydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutedcycloheteroalkyl, aryl, substituted aryl, heteroaryl or substitutedheteroaryl, or optionally R⁶⁰ and R⁶¹ together with the nitrogen atom towhich they are bonded form a cycloheteroalkyl or substitutedcycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independently hydrogen,alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁴ and R⁶⁵ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring. In some embodiments, substituentsinclude -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S, —NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN,—OCN, —SCN, —NO, —NO₂, —ONO₂, ═N₂, —N₃, —S(O)₂R⁶⁰, —OS(O₂)O⁻,—OS(O)₂R⁶⁰, —P(O)(O⁻)₂, P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —NR⁶²C(O)NR⁶⁰R⁶¹, where R⁶⁰,R⁶¹ and R⁶² are as defined above. In other embodiments, substituentsinclude -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —ONO₂,—S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰,—C(O)NR⁶⁰R⁶¹ and —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above.In still other embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰,—SR⁶⁰, —NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —ONO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹),—C(O)R⁶⁰, —C(O)OR⁶⁰ and —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as definedabove.

“Treating” or “treatment” of any disease or disorder refers, in someembodiments, to ameliorating the disease or disorder (i.e., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In other embodiments, “treating” or “treatment”refers to ameliorating at least one physical parameter, which may not bediscernible by the patient. In yet other embodiments “treating” or“treatment” refers to inhibiting the disease or disorder, eitherphysically (e.g., stabilization or eradication of a discerniblesymptom), physiologically (e.g., stabilization or eradication of aphysical parameter), or both. In still other embodiments, “treating” or“treatment” refers to delaying the onset of the disease or disorder.

“Therapeutically effective amount” means the amount of a compound that,when administered to a patient for treating or preventing a disease, issufficient to effect such treatment or prevention of the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity, and the age, weight, etc., of the patientto be treated.

Reference will now be made in detail to embodiments of the invention.While the invention will be described in conjunction with theseembodiments, it will be understood that it is not intended to limit theinvention to those preferred embodiments. To the contrary, it isintended to cover alternatives, modifications, and equivalents as may beincluded within the scope of the invention as defined by the appendedclaims.

Methods of Using O-Nitro Compounds to Treat or Prevent Abnormal CellProliferation

The present invention provides O-nitro compounds, pharmaceuticalcompositions of O-nitro compounds and methods of using O-nitro compoundsor pharmaceutical compositions thereof to treat or prevent diseasesassociated with abnormal cell proliferation.

The methods generally involve administering to a patient in need of suchtreatment or prevention a therapeutically effective amount of an O-nitrocompound or a pharmaceutically acceptable salt, hydrate, solvate orN-oxide thereof. In some embodiments, the O-nitro compound isintracellularly activated by the reducing environment of a tumor cell.In other embodiments, the patient is irradiated to activate the O-nitrocompound. Accordingly, in some embodiments, the O-nitro compounds of thepresent invention may be activated by both intracellular reduction andexternal irradiation. In these embodiments, a synergistic or additiveeffect may be observed.

O-nitro compounds are generally organic compounds substituted with oneor more O-nitro groups. Typically, O-nitro compounds have a highenthalapy of formation (i.e., decomposition of O-nitro compoundsreleases a high amount of energy). Preferably, O-nitro compounds have anenthalapy of formation that varies between about 5 kcal/mole and about150 kcal/mole, more preferably, between about 10 kcal/mole and about 110kcal/mole. The enthalapy of formation of O-nitro compounds may bereadily calculated by methods known to the skilled artisan. Accordingly,O-nitro compounds include those O-nitro compounds that decompose withexplosive force upon activation. Such compounds may be readilyidentified by those of skill in the art by calculation of the enthalapyof formation.

O-nitro compounds may also be reduced at low reduction potentials.Cyclic voltametry demonstrates that electron transfer to O-nitrocompounds occurs between about −0.1 volt and about −1.0 volt usingstandard electrodes (e.g., mercury or carbon cathode and platinum anode)and electrolyte solutions.

O-nitro compounds may contain a high density of nitro groups (i.e., thenitro groups represent a significant fraction of the overall mass of thecompound). In some embodiments, an O-nitro compound contains two nitrogroups. In other embodiments, an O-nitro compound contains three nitrogroups. In still other embodiments, an O-nitro compound contains sixnitro groups.

In some embodiments, the O-nitro compound has a ratio of nitro groups tocarbon atoms of 1:1. In other embodiments, the O-nitro compound has aratio of nitro groups to carbon atoms of 1:2.

In some embodiments, the O-nitro compound has the structure R¹—O—NO₂where R¹ is alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl,substituted heteroalkyl, fused cycloalkyl, substituted fused cycloalkyl,substituted cycloalkyl, fused cycloalkylcycloheteroalkyl, substitutedfused cycloalkylcycloheteroalkyl, cubyl, substituted cubyl, adamantyl orsubstituted adamantyl.

In other embodiments, R¹—O—NO₂ has the structure:

wherein R², R³, R⁴, R⁵ and R⁶ are independently hydrogen, alkyl orsubstituted alkyl.

In still other embodiments, R¹—O—NO₂ has the structure:

In still other embodiments, R¹—O—NO₂ has the structure:

In still other embodiments, R¹—O—NO₂ has the structure:

wherein X is —S—, —O—, —N(R⁷), —P(O)OR⁷, or —BR⁷ where R⁷ is hydrogen oralkyl.

In still other embodiments, R¹—O—NO₂ has the structure:

wherein X is —S—, —O—, —N(R⁷), —P(O)OR⁷, or —BR⁷ where R⁷ is hydrogen oralkyl.

In still other embodiments, R¹—O—NO₂ has the structure:

In still other embodiments, R¹—O—NO₂ has the structure:

In still other embodiments, R¹—O—NO₂ has the structure:

In still other embodiments, R¹—O—NO₂ has the structure:

wherein X is —S—, —O— or —N(R⁷) where R⁷ is hydrogen or alkyl.

Nitrate esters including, but not limited to, diglycerol tetranitrate,3-nitratomethyl oxetane, bis 3,3-nitrotomethyl oxetane, triethyleneglycol dinitrate, trimethylol trinitrate, pentaerythritol tetranitrate,n-butyl-2-nitratomethyl nitramine and polyglycidyl nitrate can be topractice the instant invention.

O-nitro compounds may exist in several tautomeric forms and mixturesthereof. O-nitro compounds may also include isotopically labeledcompounds where one or more atoms have an atomic mass different from theatomic mass conventionally found in nature. Examples of isotopes thatmay be incorporated into O-nitro compounds include, but are not limitedto, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O and ¹⁷O. O-nitro compounds may exist inunsolvated forms as well as solvated forms, including hydrated forms orN-oxides. In general, hydrated and solvated fowls are within the scopeof the present invention. Certain O-nitro compounds may exist inmultiple crystalline or amorphous forms. In general, all physical formsare equivalent for the uses contemplated by the present invention andare intended to be within the scope of the present invention.

O-nitro compounds may be activated by intracellular reduction. In someembodiments, O-nitro compounds are activated by intracellular reductionin hypoxic tumor cells, secondary to elevated glutathione levels (highGSH:GSSG (i.e., glutathione to glutathione disulfide ratios)) andpossibly high levels of other antioxidant enzymes in many tumor cellsand/or a median tumor cell pO₂ of less than about 10 mm Hg.

O-nitro compounds may also be activated by application of externalenergy. Methods useful for decomposing O-nitro compounds include, butare not limited to, irradiation (e.g., with x-rays, visible light,infrared irradiation), ultrasound (e.g., focused ultrasound),electrochemical reduction, heating, co-administration of free radicalinitiators (e.g., thiols), etc. In some embodiments, an O-nitro compoundis activated by photon irradiation of the patient. In other embodiments,the patient's tumor is irradiated using a linear accelerator at a doserate of about 100 cGy/min. In still other embodiments, the patient mayalso be treated with electron beam therapy, interoperative radiationtherapy, stereostatic radiosurgery and high or low dose brachytherapy.

In still other embodiments, the entire patient may be irradiated. Instill other embodiments, a portion of the patient is irradiated so thatonly the O-nitro compound localized in the irradiated portion (e.g.,tumor region) of the patient is activated. Preferably, the portion ofthe patient that is irradiated is the site of abnormal cellproliferation.

Without wishing to be bound by theory, irradiation or reduction ofO-nitro compounds may lead to formation of free radicals, such as thealkoxy radical, infra, that subsequently prevent cell replication andkill cells, presumably by interfering with DNA replication and/orreacting with cell membranes. However, other mechanisms may account forthe efficacy of O-nitro compounds in treating or preventing abnormalcell proliferation. FIG. 1 illustrates a possible mechanism that mayreflect why O-nitro compounds are effective in cancer therapy. Reductionand/or hemolytic cleavage of the O-nitro compound GLYN leads to analkoxy radical and the NO₂ radical. The NO₂ radical decomposes viafurther reduction and loss of water to NO, which is well known to thoseof skill in the pharmaceutical arts as a potent vasodilating agent. NOmay alter blood flow and permeability in pathological regions, which, inturn, can affect drug delivery. NO may also alter the degree of tissueoxygenation, which may alter the effect of radiation therapy.

O-nitro compounds may be obtained via conventional synthetic methodsdescribed in the art or are commercially available. Starting materialsuseful for preparing O-nitro compounds and intermediates thereof arecommercially available or can be prepared by well-known syntheticmethods. Other methods for synthesis of the O-nitro compounds describedherein and/or starting materials are either described in the art or willbe readily apparent to the skilled artisan.

In accordance with the invention, an O-nitro compound or apharmaceutical composition thereof is administered to a patient,preferably a human, suffering from a disease characterized by abnormalcell proliferation. The O-nitro compound and pharmaceutical compositionsthereof may be used to treat or prevent diseases characterized byabnormal cell proliferation.

Diseases characterized by abnormal cell proliferation include cancer(e.g., any vascularized tumor, solid tumors, including, but not limitedto, carcinomas of the lung, breast, ovary, stomach, pancreas, larynx,esophagus, testes, liver, parotid, bilary tract, colon, rectum, cervix,uterus, endometrium, kidney, bladder, prostrate and thyroid,lymphohematopoietic malignancies, squamous cell carcinomas,adenocarcinomas, small cell carcinomas, melanomas, gliomas,neuroblastomas, sarcomas (e.g., angiosarcomas, chondrosarcomas),diabetes, cardiovascular diseases (e.g., arteriosclerosis), inflammatorydiseases (e.g., arthritis, diabetic retinopathy, rheumatoid arthritis,neovascular glaucoma and psoriasis) and autoimmune diseases.

In other embodiments, O-nitro compounds may be used for in vitrosterilization. Biological solutions may be treated with O-nitrocompounds, which are toxic to pathogenic bacteria, viruses and cells.This process can also be catalyzed by the application of external energysuch as light and heat.

Further, in certain embodiments, an O-nitro compound and/orpharmaceutical compositions thereof are administered to a patient,preferably a human, as a preventative measure against various diseasesor disorders characterized by abnormal cell proliferation. Thus, O-nitrocompounds and/or pharmaceutical compositions thereof may be administeredas a preventative measure to a patient having a predisposition for adisease characterized by abnormal cell proliferation. Accordingly,O-nitro compounds and/or pharmaceutical compositions thereof may be usedfor the prevention of one disease or disorder and concurrently treatinganother (e.g., preventing arthritis while treating cancer).

The suitability of O-nitro compounds and/or pharmaceutical compositionsthereof in treating or preventing various diseases or disorderscharacterized by abnormal cell proliferation may be determined bymethods described in the art.

Therapeutic/Prophylactic Administration

O-nitro compounds and/or pharmaceutical compositions thereof may beadvantageously used in human medicine. As previously described, O-nitrocompounds and/or pharmaceutical compositions thereof are useful for thetreatment or prevention of various diseases or disorders.

When used to treat or prevent the above disease or disorders, O-nitrocompounds and/or pharmaceutical compositions thereof may be administeredor applied singly, or in combination with other agents. O-nitrocompounds and/or pharmaceutical compositions thereof may also beadministered or applied singly or in combination with otherpharmaceutically active agents (e.g., other anti-cancer agents),including other O-nitro compounds and/or pharmaceutical compositionsthereof.

The current invention provides methods of treatment and prophylaxis byadministration to a patient of a therapeutically effective amount of anO-nitro compound and/or pharmaceutical composition thereof The patientis preferably a mammal and, most preferably, is a human.

O-nitro compounds and/or pharmaceutical compositions thereof may beadministered orally. O-nitro compounds and/or pharmaceuticalcompositions thereof may also be administered by any other convenientroute, for example, by infusion or bolus injection, by absorptionthrough epithelial or mucocutaneous linings (e.g., oral mucosa, rectaland intestinal mucosa, etc.). Administration can be systemic or local.Various delivery systems are known (e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc.) that can be used toadminister an O-nitro compound and/or pharmaceutical compositionthereof. Methods of administration include, but are not limited to,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, oral, sublingual, intranasal, intracerebral,intravaginal, transdermal, rectally, by inhalation, or topically,particularly to the ears, nose, eyes or skin. The preferred mode ofadministration is left to the discretion of the practitioner and willdepend in part upon the site of the medical condition. In mostinstances, administration will result in the release of O-nitrocompounds and/or pharmaceutical compositions thereof into thebloodstream.

In specific embodiments, it may be desirable to administer one or moreO-nitro compounds and/or pharmaceutical compositions thereof locally tothe area in need of treatment. This may be achieved, for example, andnot by way of limitation, by local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, the implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes orfibers. In some embodiments, administration can be by direct injectionat the site (or former site) of the disease or disorder.

In certain embodiments, it may be desirable to introduce one or moreO-nitro compounds and/or pharmaceutical compositions thereof into thecentral nervous system by any suitable route, includingintraventricular, intrathecal and epidural injection. Intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir.

O-nitro compounds and/or pharmaceutical compositions thereof may also beadministered directly to the lung by inhalation. For administration byinhalation, O-nitro compounds and/or pharmaceutical compositions thereofmay be conveniently delivered to the lung by a number of differentdevices. For example, a Metered Dose Inhaler (“MDI”), which utilizescanisters that contain a suitable low boiling propellant (e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or any other suitable gas) maybe used to deliver O-nitro compounds and/or pharmaceutical compositionsthereof directly to the lung.

Alternatively, a Dry Powder Inhaler (“DPI”) device may be used toadminister an O-nitro compound and/or pharmaceutical composition thereofto the lung. DPI devices typically use a mechanism such as a burst ofgas to create a cloud of dry powder inside a container, which may thenbe inhaled by the patient and are well known in the art. A popularvariation is the multiple dose DPI (“MDDPI”) system, which allows forthe delivery of more than one therapeutic dose. MDDPI devices arecommercially available from a number of pharmaceutical companies (e.g.,Schering Plough, Madison, N.J.). For example, capsules and cartridges ofgelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of an O-nitro compound and/or pharmaceuticalcomposition thereof and a suitable powder base such as lactose or starchfor these systems.

Another type of device that may be used to deliver an O-nitro compoundand/or pharmaceutical composition thereof to the lung is a liquid spraydevice supplied, for example, by Aradigm Corporation, Hayward, Calif.Liquid spray systems use extremely small nozzle holes to aerosolizeliquid drug formulations that may then be directly inhaled into thelung.

In some embodiments, a nebulizer is used to deliver an O-nitro compoundand/or pharmaceutical composition thereof to the lung. Nebulizers createaerosols from liquid drug formulations by using, for example, ultrasonicenergy to form fine particles that may be readily inhaled (see, e.g.,Raleigh et al., British J. Cancer, 1999, 80, Suppl. 2, 96). Examples ofnebulizers include devices supplied by Sheffield Pharmaceuticals, St.Louis, Mo. (Armer et al., U.S. Pat. No. 5,954,047; van der Linden etal., U.S. Pat. No. 5,950,619; van der Linden et al., U.S. Pat. No.5,970,974), and Batelle Pulmonary Therapeutics, Columbus, Ohio.

In some embodiments, an electrohydrodynamic (“EHD”) aerosol device isused to deliver an O-nitro compound and/or pharmaceutical compositionthereof to the lung of a patient. EHD aerosol devices use electricalenergy to aerosolize liquid drug solutions or suspensions (see, e.g.,Noakes et al., U.S. Pat. No. 4,765,539). The electrochemical propertiesof the formulation may be important parameters to optimize whendelivering an O-nitro compound and/or pharmaceutical composition thereofto the lung with an EHD aerosol device and such optimization isroutinely performed by one of skill in the art. EHD aerosol devices maymore efficiently deliver drugs to the lung than existing pulmonarydelivery technologies.

In other embodiments, an O-nitro compound and/or pharmaceuticalcomposition thereof can be delivered in a vesicle, in particular, aliposome (e.g., Langer, 1990, Science 249:1527-1533; Treat et al., in“Liposomes in the Therapy of Infectious Disease and Cancer,”Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).

In still other embodiments, an O-nitro compound and/or pharmaceuticalcomposition thereof can be delivered via sustained release systems,preferably oral sustained release systems. In some embodiments, a pumpmay be used (e.g., Langer, supra, Sefton, 1987, CRC Crit. Ref Biomed.Eng. 14:201; Saudek et al., 1989, N. Engl. J. Med. 321:574).

In still other embodiments, polymeric materials can be used (e.g.,“Medical Applications of Controlled Release,” Langer and Wise (eds.),CRC Press, Boca Raton, Fla. (1974); “Controlled Drug Bioavailability,”Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Langer et al., 1983, J. Macromol. Sci. Rev. Macromol. Chem.23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

In other embodiments, polymeric materials are used for oral sustainedrelease delivery. Polymers include, but are not limited to, sodiumcarboxymethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose and hydroxyethylcellulose (most preferred,hydroxypropyl methylcellulose). Other cellulose ethers have beendescribed in the art (Alderman, Int. J. Pharm. Tech. & Prod. Mfr. 1984,5(3) 1-9). Factors affecting drug release are well known to the skilledartisan and have been described in the art (Bamba et al., Int. J. Pharm.1979, 2:307).

In still other embodiments, enteric-coated preparations can be used fororal sustained release administration. Coating materials include, butare not limited to, polymers with a pH-dependent solubility (i.e.,pH-controlled release), polymers with a slow or pH-dependent rate ofswelling, dissolution or erosion (i.e., time-controlled release),polymers that are degraded by enzymes (i.e., enzyme-controlled release)and polymers that form firm layers that are destroyed by an increase inpressure (i.e., pressure-controlled release).

In still other embodiments, osmotic delivery systems are used for oralsustained release administration (Verma et al., Drug Dev. Ind. Pharm.,2000, 26:695-708). In other embodiments, OROS® osmotic devices are usedfor oral sustained release delivery devices (Theeuwes et al., U.S. Pat.No. 3,845,770; Theeuwes et al., U.S. Pat. No. 3,916,899).

In other embodiments, a controlled-release system can be placed inproximity of the target of the O-nitro compound and/or pharmaceuticalcomposition, thus requiring only a fraction of the systemic dose (e.g.,Goodson, in “Medical Applications of Controlled Release,” supra, vol. 2,pp. 115-138 (1984)). Other controlled-release systems previously mayalso be used (Langer, 1990, Science 249:1527-1533).

Pharmaceutical Compositions

The present pharmaceutical compositions typically contain atherapeutically effective amount of one or more O-nitro compounds,preferably in purified form, together with a suitable amount of apharmaceutically acceptable vehicle, so as to provide the form forproper administration to a patient. When administered to a patient, theO-nitro compound and pharmaceutically acceptable vehicles are preferablysterile. Water is a preferred vehicle when the O-nitro compound isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid vehicles, particularlyfor injectable solutions. Suitable pharmaceutical vehicles also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present pharmaceutical compositions, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. In addition, auxiliary, stabilizing,thickening, lubricating and coloring agents may be used.

Pharmaceutical compositions comprising an O-nitro compound may bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries, which facilitateprocessing of compounds into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The present pharmaceutical compositions can take the form of solutions,suspensions, emulsion, tablets, pills, pellets, capsules, capsulescontaining liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for use. In some embodiments, the pharmaceuticallyacceptable vehicle is a capsule (e.g., Grosswald et al., U.S. Pat. No.5,698,155). A general discussion of the preparation of pharmaceuticalcompositions may be found in Remington, “The Science and Practice ofPharmacy,” 19th Edition.

For topical administration, an O-nitro compound may be formulated assolutions, gels, ointments, creams, suspensions, etc., as is well knownin the art.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration. Systemic formulationsmay be made in combination with a further active agent that improvesmucociliary clearance of airway mucus or reduces mucous viscosity. Theseactive agents include, but are not limited to, sodium channel blockers,antibiotics, N-acetyl cysteine, homocysteine and phospholipids.

In some embodiments, O-nitro compounds are formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, O-nitro compoundsare solutions in sterile isotonic aqueous buffer for intravenousadministration. For injection, O-nitro compounds may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. When necessary, the pharmaceuticalcompositions may also include a solubilizing agent. Pharmaceuticalcompositions for intravenous administration may optionally include alocal anesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a lyophilized powderor water-free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. When theO-nitro compounds are administered by infusion, it can be dispensed, forexample, with an infusion bottle containing sterile pharmaceutical gradewater or saline. When the O-nitro compound is administered by injection,an ampoule of sterile water for injection or saline can be provided sothat the ingredients may be mixed prior to administration.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Pharmaceutical compositions for oral delivery may be in the form oftablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Orallyadministered pharmaceutical compositions may contain one or moreoptional agents, for example, sweetening agents such as fructose,aspartame or saccharin; flavoring agents such as peppermint, oil ofwintergreen, or cherry; coloring agents; and preserving agents, toprovide a pharmaceutically palatable preparation. Moreover, when intablet or pill form, the pharmaceutical compositions may be coated todelay disintegration and absorption in the gastrointestinal tract,thereby providing a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for orally administered compounds. Inthese later platforms, fluid from the environment surrounding thecapsule is imbibed by the driving compound, which swells to displace theagent or agent composition through an aperture. These delivery platformscan provide an essentially zero order delivery profile as opposed to thespiked profiles of immediate release formulations. A time delay materialsuch as glycerol monostearate or glycerol stearate may also be used.Oral compositions can include standard vehicles such as mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such vehicles are preferably of pharmaceuticalgrade.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,saline, alkyleneglycols (e.g., propylene glycol), polyalkylene glycols(e.g., polyethylene glycol), oils, alcohols, slightly acidic buffersbetween pH 4 and pH 6 (e.g., acetate, citrate, or ascorbate at betweenabout 5.0 mM to about 50.0 mM), etc. Additionally, flavoring agents,preservatives, coloring agents, bile salts, acylcarnitines and the likemay be added.

For buccal administration, the pharmaceutical compositions may take theform of tablets, lozenges, etc., formulated in a conventional manner.

Liquid drug formulations suitable for use with nebulizers and liquidspray devices and EHD aerosol devices typically include an 0-nitrocompound with a pharmaceutically acceptable vehicle. In someembodiments, the pharmaceutically acceptable vehicle is a liquid such asalcohol, water, polyethylene glycol or a perfluorocarbon. Optionally,another material may be added to alter the aerosol properties of thesolution or suspension of compounds. This material may be a liquid suchas an alcohol, glycol, polyglycol or a fatty acid. Other methods offormulating liquid drug solutions or suspension suitable for use inaerosol devices are known to those of skill in the art (see, e.g.,Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No. 5,556,611).

An O-nitro compound may also be formulated in rectal or vaginalpharmaceutical compositions such as suppositories or retention enemas,e.g., containing conventional suppository bases such as cocoa butter orother glycerides.

In addition to the formulations described previously, an O-nitrocompound may also be formulated as a depot preparation. Such long-actingformulations may be administered by implantation (for example,subcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, an O-nitro compound may be formulated with suitablepolymeric or hydrophobic materials (e.g., as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, such as a sparingly soluble salt.

When an O-nitro compound is acidic or basic, it may be included in anyof the above-described formulations as the free acid or free base, apharmaceutically acceptable salt, a solvate or hydrate. Pharmaceuticallyacceptable salts substantially retain the activity of the free acid orbase, may be prepared by reaction with bases or acids and tend to bemore soluble in aqueous and other protic solvents than the correspondingfree acid or base form.

Doses

An O-nitro compound and/or pharmaceutical composition thereof, willgenerally be used in an amount effective to achieve the intendedpurpose. For use in treating or preventing the above diseases ordisorders, the O-nitro compound and/or pharmaceutical compositionsthereof are administered or applied in a therapeutically effectiveamount.

The amount of an O-nitro compound and/or pharmaceutical compositionthereof that will be effective in the treatment of a particular disorderor condition disclosed herein will depend on the nature of the disorderor condition, and can be determined by standard clinical techniquesknown in the art. In addition, in vitro or in vivo assays may optionallybe employed to help identify optimal dosage ranges. The amount of anO-nitro compound and/or pharmaceutical composition thereof administeredwill, of course, be dependent on, among other factors, the subject beingtreated, the weight of the subject, the severity of the affliction, themanner of administration and the judgment of the prescribing physician.

For example, the dosage may be delivered in a pharmaceutical compositionby a single administration, by multiple applications or controlledrelease. Dosing may be repeated intermittently, may be provided alone orin combination with other drugs and may continue as long as required foreffective treatment of the disease state or disorder.

Suitable dosage ranges for oral administration are dependent on theefficiency of radiosensitization, but are generally about 0.001 mg toabout 100 mg of the O-nitro compound per kg body weight. Dosage rangesmay be readily determined by methods known to the artisan of ordinaryskill.

Suitable dosage ranges for intravenous (i.v.) administration are about0.01 mg to about 100 mg per kg/body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 mg/kg body weight toabout 1 mg/kg body weight. Suppositories generally contain about 0.01milligram to about 50 milligrams of an O-nitro compound per kg/bodyweight and comprise active ingredient in the range of about 0.5% toabout 10% by weight. Recommended dosages for intradermal, intramuscular,intraperitoneal, subcutaneous, epidural, sublingual or intracerebraladministration are in the range of about 0.001 mg to about 200 mg perkg/body weight. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems. Such animalmodels and systems are well known in the art.

The O-nitro compounds may be assayed in vitro and in vivo, for thedesired therapeutic or prophylactic activity, prior to use in humans.For example, in vitro assays can be used to determine whetheradministration of a specific O-nitro compound or a combination ofO-nitro compounds is preferred. The O-nitro compound may also bedemonstrated to be effective and safe using animal model systems.

Preferably, a therapeutically effective dose of an O-nitro compoundand/or pharmaceutical composition thereof described herein will providetherapeutic benefit without causing substantial toxicity. Toxicity ofO-nitro compounds and/or pharmaceutical compositions thereof may bedetermined using standard pharmaceutical procedures and may be readilyascertained by the skilled artisan. The dose ratio between toxic andtherapeutic effect is the therapeutic index. An O-nitro compound and/orpharmaceutical composition thereof will preferably exhibit particularlyhigh therapeutic indices in treating disease and disorders characterizedby aberrant abnormal cell proliferation. The dosage of an O-nitrocompound and/or pharmaceutical composition thereof described herein willpreferably be within a range of circulating concentrations that includean effective dose with little or no toxicity.

Combination Therapy

In certain embodiments, 0-nitro compounds and/or pharmaceuticalcompositions thereof can be used in combination therapy with at leastone other therapeutic agent. The O-nitro compound and/or pharmaceuticalcomposition thereof and the therapeutic agent can act additively or,more preferably, synergistically. In some embodiments, an O-nitrocompound and/or a pharmaceutical composition thereof is administeredconcurrently with the administration of another therapeutic agent. Inother embodiments, an O-nitro compound and/or pharmaceutical compositionthereof is administered prior or subsequent to administration of anothertherapeutic agent.

In particular, in some embodiments, O-nitro compounds and/orpharmaceutical compositions thereof can be used in combination therapywith other chemotherapeutic agents (e.g., alkylating agents (e.g.,nitrogen mustards (e.g., cyclophosphamide, ifosfamide, mechlorethamine,melphalen, chlorambucil, hexamethylmelamine, thiotepa), alkyl sulfonates(e.g., busulfan), nitrosoureas, or triazines)), antimetabolites (e.g.,folic acid analogs, pyrimidine analogs (e.g., fluorouracil, floxuridine,cytosine arabinoside, etc.)), purine analogs (e.g., mercaptopurine,thiogunaine, pentostatin, etc.), natural products (e.g., vinblastine,vincristine, etoposide, tertiposide, dactinomycin, daunorubicin,doxurubicin, bleomycin, mithramycin, mitomycin C, L-asparaginase,interferon alpha), platinum coordination complexes (e.g., cis-platinum,carboplatin, etc.), apoptosis-inducing agents, glutathione-depletingagents or other agents that can alter the redox status of the cell.Those of skill in the art will appreciate that O-nitro compounds mayalso be used in concurrent combination therapy with both thechemotherapeutic agents listed above and radiotherapy.

Therapeutic Kits

The current invention also provides therapeutic kits comprising O-nitrocompounds and/or pharmaceutical compositions thereof. The therapeutickits may also contain other compounds (e.g., chemotherapeutic agents,natural products, apoptosis-inducing agents, etc.) or pharmaceuticalcompositions thereof.

Therapeutic kits may have a single container that contains an O-nitrocompound and/or pharmaceutical compositions thereof with or withoutother components (e.g., other compounds or pharmaceutical compositionsof these other compounds) or may have a distinct container for eachcomponent. In some embodiments, therapeutic kits include an O-nitrocompound and/or a pharmaceutical composition thereof packaged for use incombination with the co-administration of a second compound (preferably,a chemotherapeutic agent, a natural product, an apoptosis-inducingagent, etc.) or a pharmaceutical composition thereof. The components ofthe kit may be pre-complexed or each component may be in a separatedistinct container prior to administration to a patient.

The components of the kit may be provided in one or more liquidsolutions, such as an aqueous solution or a sterile aqueous solution.The components of the kit may also be provided as solids, which may beconverted into liquids by addition of suitable solvents, which may beprovided in another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid.

Preferably, a therapeutic kit will contain apparatus (e.g., one or moreneedles, syringes, eye droppers, pipettes, etc.), which enablesadministration of the components of the kit.

EXAMPLES

The invention is further defined by reference to the following examples,which describe in detail experiments that, inter alia, demonstrate theeffectiveness of O—NO₂ compounds in tumor cell therapy. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, may be practiced without departing from thescope.

Example 1 Chemosensitization Effect of GLYN in Combination withCis-Platin on SCC VII Tumor Growth

Mice were implanted subcutaneously with a murine squamous cell carcinomaSCC VII tumor cells. When tumors grew to 100-150 mm³ (12 days afterimplantation), mice with tumors were treated with a single dose of GLYN(100 mg/kg or 300 mg/kg), cis-platin (CDDP, 2 or 5 mg/kg) or incombination. Tumors were measured immediately before treatment and threetimes a week thereafter.

As can be seen in FIG. 2 and Table 1, GLYN at doses of 100 mg/kg or 300mg/kg significantly enhanced the responses of SCC VII tumors to thetreatment with cis-platin. Cis-platin alone at 5 mg/kg inhibited the 4×tumor growth delay time by 1.1 days. When combined with GLYN, the 4×tumor growth delay times increased to 2.8 days and 7.2 days for GLYN 100mg/kg and 300 mg/kg, respectively, representing a two- to seven-foldincrease (p<0.01 compared with cis-platin alone). The systemic toxicityindicated by the body weight loss was moderate and mice were recoveredone week after treatment (data not shown).

TABLE 1 Comparison of SCC VII tumor growth time in mice treated withGLYN and cis-platin Num- ber of 4x TGT TGD P value (t-test Mice (day)(day) Control GLYN CDDP Control 5 3.0 ± 0.4 GLYN 5 3.6 ± 0.5 0.5 ± 0.50.1 300 mg CDDP 5 4.1 ± 0.4 1.1 ± 0.4 <0.01 0.1 5 mg GLYN 6 5.8 ± 0.72.8 ± 0.7 <0.01 <0.01 <0.01 100 mg + CDDP 5 mg GLYN 7 10.2 ± 3.1  7.2 ±3.1 <0.01 <0.01 <0.01 300 mg + CDDP 5 mg * 4x TGT (tumor growth time):tumor volume quadrupling time. ** TGD: tumor growth delay time, i.e., 4xTGT of treated tumors minus the mean 4x TGT of control tumors.

Example 2 Radiosensitization Effect of GLYN and SG on SCC VII TumorGrowth

Mice were implanted subcutaneously with SCC VII tumor cells. When tumorsreached 100-150 mm³, mice with tumors were treated with a single dose ofGLYN (200 mg/kg), sodium glycididazole (SG, 400 mg/kg), 7 Gy radiation,or in combination. Tumors were measured immediately before treatment andthree times a week thereafter.

As shown in FIG. 3, a single dose of 7 Gy radiation (RT7Gy) inhibitedthe 4× tumor growth by 0.9±1.4 days. The combined therapy of radiationand GLYN or SG inhibited the 4× tumor growth by 4.4 and 3.3 days (p<0.05compared with radiation alone), respectively. There was no statisticallysignificant difference in the 4× tumor growth delay time between thecombination therapy of radiation plus GLYN and radiation plus SG(p=0.2).

Example 3 Production of Nitric Oxide (NO) in Tumor Cells by GLYN

SCC VII tumor cells were grown in 96-well plates overnight at 37° C. Afluorescent probe DAF-FM diacetate was added at a concentration of 10 μMfor 1 hour and then washed out. GLYN was added in the growth media atconcentrations of 0.1 mM, 1 mM or 10 mM. The green fluorescence wasmeasured at 0 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 6 hoursand 24 hours after addition of GLYN using a microplatespectrofluorometer with an excitation at 495 nm and an emission at 515nm (see FIGS. 4 a and 4 b).

Example 4 Production of Nitric Oxide in Tumor Cells by GLYN andRadiation

SCC VII tumor cells were grown in 96-well plates overnight at 37° C. Afluorescent probe DAF-FM diacetate was added at a concentration of 10 μMfor 1 hour and then washed out. GLYN was added in the growth media atconcentrations of 0.1 mM, 1 mM or 10 mM. Cells were immediatelyirradiated with radiation doses of 2 or 10 Gy. The green fluorescencewas measured at 0 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 6hours and 24 hours after addition of GLYN using a microplatespectrofluorometer with an excitation at 495 nm and an emission at 515nm. FIGS. 5 a-5 d show the production of nitric oxide in SCC VII cellsat selected times after exposure to GLYN and radiation. Radiation aloneproduced a low level of NO in SCC VII tumor cells. When combined withGLYN, the intracellular level of NO slightly increased.

Finally, it should be noted that there are alternative ways ofimplementing the present invention. Accordingly, the present embodimentsare to be considered as illustrative and not restrictive, and theinvention is not to be limited to the details given herein, but may bemodified within the scope and equivalents of the appended claims. Allreferences, publications and patents cited herein are incorporated byreference in their entirety.

What is claimed is:
 1. A pharmaceutical composition comprising glycidylnitrate or a pharmaceutically acceptable salt, hydrate or solvatethereof, a pharmaceutically acceptable vehicle, and cis-platin.
 2. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition comprises a salt of glycidyl nitrate.
 3. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition comprisesa therapeutically effective amount of glycidyl nitrate or apharmaceutically acceptable salt, hydrate or solvate thereof.
 4. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition comprises from about 0.001 mg to about 100 mg of theglycidyl nitrate or a pharmaceutically acceptable salt, hydrate orsolvate thereof per kg body weight.